1. Technical Field
The subject invention relates to the identification and isolation of a gene that encodes an enzyme (i.e., human xcex945-desaturase) involved in the synthesis of polyunsaturated fatty acids and to uses thereof. In particular, xcex945-desaturase catalyzes the conversion of, for example, dihomo-xcex3-linolenic acid (DGLA) to arachidonic acid (AA) and (n-3)-eicosatetraenoic acid (20:4n-3) to eicosapentaenoic acid (20:5n-3). The converted product may then be utilized as a substrate in the production of other polyunsaturated fatty acids (PUFAs). The product or other polyunsaturated fatty acids may be added to pharmaceutical compositions, nutritional composition, animal feeds as well as other products such as cosmetics.
2. Background Information
Desaturases are critical in the production of long-chain polyunsaturated fatty acids which have many important functions. For example, PUFAs are important components of the plasma membrane of a cell, where they are found in the form of phospholipids. They also serve as precursors to mammalian prostacyclins, eicosanoids, leukotrienes and prostaglandins. Additionally, PUFAs a re necessary for the proper development of the developing infant brain as well as for tissue formation and repair. In view of the biological significance of PUFAs, attempts are being made to produce them, as well as intermediates leading to their production, in an efficient manner.
A number of enzymes are involved in PUFA biosynthesis including xcex945-desaturase (see FIG. 11). For example, elongase (elo) catalyzes the conversion of xcex3-linolenic acid (GLA) to dihomo -xcex3-linolenic acid (DGLA) and of stearidonic acid (18:4n-3) to (n-3)-eicosatetraenoic acid (20:4n-3). Linoleic acid (LA, 18:2-xcex949, 12 or 18:2n-6) is produced from oleic acid (18:1-xcex949) by a xcex9412-desaturase. GLA (18:3-xcex946, 9, 12) is produced from linoleic acid by a xcex946-desaturase.
It must be noted that animals cannot desaturate beyond the xcex949 position and therefore cannot convert oleic acid into linoleic acid. Likewise, xcex1-linolenic acid (ALA, 18:3-xcex949, 12, 15) cannot be synthesized by mammals. However, xcex1-linolenic acid can be converted to stearidonic acid (STA, 18:4-xcex946, 9, 12, 15) by a xcex946-desaturase (see PCT publication WO 96/13591 and The Faseb Journal, Abstracts, Part I, Abstract 3093, page A532 (Experimental Biology 98, San Francisco, Calif., Apr. 18-22, 1998) see also U.S. Pat. No. 5,552,306), followed by elongation to (n-3)-eicosatetraenoic acid (20:4-xcex948, 11, 14, 17) in mammals and algae. This polyunsaturated fatty acid (i.e., 20:4-xcex948, 11, 14, 17) can then be converted to eicosapentaenoic acid (EPA, 20:5-xcex945, 8, 11, 14, 17) by a xcex945-desaturase, such as that of the present invention. Other eukaryotes, including fungi and plants, have enzymes which desaturate at carbon 12 (see PCT publication WO 94/11516 and U.S. Pat. No. 5,443,974) and carbon 15 (see PCT publication WO 93/11245). The major polyunsaturated fatty acid of animals therefore are either derived from diet and/or from desaturation and elongation of linoleic acid or xcex1-linolenic acid. In view of these difficulties, it is of significant interest to isolate genes involved in PUFA synthesis from species that naturally produce these fatty acids and to express these genes in a microbial, plant, or animal system which can be altered to provide production of commercial quantities of one or more PUFAs. One of the most important long chain PUFAs, noted above, is arachidonic acid (AA). AA is found in filamentous fungi and can also be purified from mammalian tissues including the liver and adrenal glands. As noted above, AA production from dihomo -xcex3-linolenic acid is catalyzed by a xcex945-desaturase. EPA is another important long-chain PUFA. EPA is found in fungi and also in marine oils. As noted above, EPA is produced from (n-3)-eicosatetraenoic acid and is catalyzed by a xcex945-desaturase.
In view of the above discussion, there is a definite need for the xcex945-desaturase enzyme, the gene encoding this enzyme, as well as recombinant methods of producing this enzyme. Additionally, a need exists for oils containing levels of PUFAs beyond those naturally present as well as those enriched in novel PUFAs. Such oils can only be made by isolation and expression of the xcex945-desaturase gene.
All U.S. patents and publications referred to herein are hereby incorporated in their entirety by reference.
The present invention includes an isolated nucleotide sequence corresponding to or complementary to at least about 50% of the nucleotide sequence shown in SEQ ID NO:1 (FIG. 12). The isolated nucleotide sequence may be represented by SEQ ID NO:1. These sequences may encode a functionally active desaturase which utilizes a polyunsaturated fatty acid as a substrate. The sequences may be derived from a mammal such as, for example, a human.
The present invention also includes purified proteins encoded by the nucleotide sequences referred to above. Additionally, the present invention includes a purified polypeptide which desaturates polyunsaturated fatty acids at carbon 5 and has at least about 50% amino acid similarity to the amino acid sequence of the purified proteins referred to directly above.
Furthermore, the present invention also encompasses a method of producing a human xcex945-desaturase. This method comprises the steps of: a) isolating the nucleotide sequence represented by SEQ ID NO:1 (FIG. 12); b) constructing a vector comprising: i) the isolated nucleotide sequence operably linked to ii) a promoter; and c) introducing the vector into a host cell under time and conditions sufficient for expression of the human xcex945-desaturase. The host cell may be, for example, a eukaryotic cell or a prokaryotic cell. In particular, the prokaryotic cell may be, for example, E. coli, cyanobacteria or B. subtilis. The eukaryotic cell may be, for example, a mammalian cell, an insect cell, a plant cell or a fungal cell (e.g., a yeast cell such as Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida spp., Lipomyces starkey, Yarrowia lipolytica, Kluyveromyces spp., Hansenula Spp., Trichoderma spp. or Pichia spp.).
Additionally, the present invention also encompasses a vector comprising: a) a nucleotide sequence as represented by SEQ ID NO:1 (FIG. 12) operably linked to b) a promoter. The invention also includes a host cell comprising this vector. The host cell may be, for example, a eukaryotic cell or a prokaryotic cell. Suitable eukaryotic cells and prokaryotic cells are as defined above.
Moreover, the present invention also includes a plant cell, plant or plant tissue comprising the above vector, wherein expression of the nucleotide sequence of the vector results in production of a polyunsaturated fatty acid by the plant cell, plant or plant tissue. The polyunsaturated fatty acid may be, for example, selected from the group consisting of AA and EPA. The invention also includes one or more plant oils or acids expressed by the above plant cell, plant or plant tissue.
Additionally, the present invention also encompasses a transgenic plant comprising the above vector, wherein expression of the nucleotide sequence of the vector results in production of a polyunsaturated fatty acid in seeds of the transgenic plant.
Also, the invention includes a mammalian cell comprising the above vector wherein expression of the nucleotide sequence of the vector results in production of altered levels of AA or EPA when the cell is grown in a culture media comprising a fatty acid selected from the group consisting of an essential fatty acid, LA and ALA.
It should also be noted that the present invention encompasses a transgenic, non-human mammal whose genome comprises a DNA sequence encoding a human xcex945-desaturase operably linked to a promoter. The DNA sequence may be represented by SEQ ID NO:1 (FIG. 12). Additionally, the present invention includes a fluid (e.g., milk) produced by the transgenic, non-human mammal wherein the fluid comprises a detectable level of at least human xcex945-desaturase.
Additionally, the present invention includes a method (i.e., xe2x80x9cfirstxe2x80x9d method) for producing a polyunsaturated fatty acid comprising the steps of: a) isolating the nucleotide sequence represented by SEQ ID NO:1 (FIG. 12); b) constructing a vector comprising the isolated nucleotide sequence; c) introducing the vector into a host cell under time and conditions sufficient for expression of the human xcex945-desaturase enzyme; and d) exposing the expressed human xcex945-desaturase enzyme to a substrate polyunsaturated fatty acid in order to convert the substrate to a product polyunsaturated fatty acid. The substrate polyunsaturated fatty acid may be, for example, DGLA or 20:4n-3 and the product polyunsaturated fatty acid may be, for example, AA or EPA, respectively. This method may further comprise the step of exposing the product polyunsaturated fatty acid to an elongase in order to convert the product polyunsaturated fatty acid to another polyunsaturated fatty acid (i.e., xe2x80x9csecondxe2x80x9d method). In this method containing the additional step (i.e., xe2x80x9csecondxe2x80x9d method), the product polyunsaturated fatty acid may be, for example, AA or EPA, and the xe2x80x9canotherxe2x80x9d polyunsaturated fatty acid may be adrenic acid or (n-3)-docosapentaenoic acid, respectively. The method containing the additional step may further comprise a step of exposing the another polyunsaturated fatty acid to an additional desaturase in order to convert the another polyunsaturated fatty acid to a final polyunsaturated fatty acid (i.e., xe2x80x9cthirdxe2x80x9d method). The final polyunsaturated fatty acid may be, for example, (n-6)-docosapentaenoic acid or docosahexaenoic (DHA) acid.
The present invention also encompasses a nutritional composition comprising at least one polyunsaturated fatty acid selected from the group consisting of the product polyunsaturated fatty acid produced according to the xe2x80x9cfirstxe2x80x9d method, another polyunsaturated fatty acid produced according to the xe2x80x9csecondxe2x80x9d method, and the final polyunsaturated fatty acid produced according to the xe2x80x9cthirdxe2x80x9d method. The product polyunsaturated fatty acid may be, for example, AA or EPA. The another polyunsaturated fatty acid may be, for example, adrenic acid or (n-3)-docosapentaenoic acid. The final polyunsaturated fatty acid may be, for example, (n-6)-docosapentaenoic acid or DHA. This nutritional composition, may be, for example, an infant formula, a dietary supplement or a dietary substitute and may be administered to a human or to an animal. It may be administered enterally or parenterally. The nutritional composition may further comprise at least one macronutrient selected from the group consisting of coconut oil, soy oil, canola oil, monoglycerides, diglycerides, glucose, edible lactose, electrodialysed whey, electrodialysed skim milk, milk whey, soy protein, and protein hydrolysates. Additionally, the composition may further comprise at least one vitamin selected from the group consisting of Vitamins A, C, D, E, and B complex and at least one mineral selected from the group consisting of calcium magnesium, zinc, manganese, sodium, potassium, phosphorus, copper, chloride, iodine, selenium and iron.
Furthermore, the present invention also includes a a pharmaceutical composition comprising 1) at least one polyunsaturated fatty acid selected from the group consisting of the product polyunsaturated fatty acid produced according to the xe2x80x9cfirstxe2x80x9d method, the another polyunsaturated fatty acid produced according to the xe2x80x9csecondxe2x80x9d method, and the final polyunsaturated fatty acid produced according to the xe2x80x9cthirdxe2x80x9d method and 2) a pharmaceutically acceptable carrier. Again, the pharmaceutical composition may be administered to a human or to an animal. The composition may further comprise an element selected from the group consisting of a vitamin, a mineral, a carbohydrate, an amino acid, a free fatty acid, a phospholipid, an antioxidant, and a phenolic compound.
Additionally, the present invention includes an animal feed comprising at least one polyunsaturated fatty acid selected from the group consisting of the product polyunsaturated fatty acid produced according to the first method, the another polyunsaturated fatty acid produced according to the second method and the final polyunsaturated fatty acid produced according to the third method. The product polyunsaturated fatty acid may be, for example, AA or EPA. The another polyunsaturated fatty acid may be, for example, adrenic acid or (n-3)-docosapentaenoic acid. The final polyunsaturated fatty acid may be, for example,(n-6)-docosapentaenoic acid or DHA.
Moreover, the present invention also includes a cosmetic comprising a polyunsaturated fatty acid selected from the group consisting of the product polyunsaturated fatty acid produced according to the first method, the another polyunsaturated fatty acid produced according to the second method, and the final polyunsaturated fatty acid produced according to the third method.
Additionally, the present invention encompasses a method of preventing or treating a condition caused by insufficient intake of polyunsaturated fatty acids comprising administering to the patient the nutritional composition of above in an amount sufficient to effect prevention or treatment.